Antibody-Based Proteomic Analysis of Apoptosis Signaling
Reagents that assess activation of apoptosis and associated signaling pathways are critical for greater understanding of the molecular basis of programmed cell death. The advent of proteomic technologies to probe these events allows monitoring of hundreds to thousands of proteins, as well as sites of posttranslational modification involved in apoptosis at one time. This view of apoptosis at a network level is a powerful tool in studying known apoptotic pathways, as well as elucidating novel signaling events that affect or are affected by apoptotic signaling. The following is a detailed method for successful proteomic profi...
Source: Springer protocols feed by Pharmacology/Toxicology - December 31, 2015 Category: Drugs & Pharmacology Source Type: news

Automated Ratio Imaging Using Nuclear-Targeted FRET Probe-Expressing Cells for Apoptosis Detection
In recent years, innovative bioassays have been designed to detect intracellular caspase activation as a reliable read-out of apoptotic activity of bioactive compounds. Most anticancer drugs target cells by triggering caspase dependent protein cleavage, culminating in apoptotic cell death. Therefore, detection of caspase activation has been recognized as one of the best approaches for detecting cancer cell death as compared to assaying the ill-defined general cytotoxic activity that often manifests with off-target side effects. Among the available methods of detection, those with cells stably expressing FRET-based fluoresc...
Source: Springer protocols feed by Pharmacology/Toxicology - December 31, 2015 Category: Drugs & Pharmacology Source Type: news

FRET-Based Measurement of Apoptotic Caspase Activities by High-Throughput Screening Flow Cytometry
Unwanted and excessive apoptosis contributes to various degenerative diseases, and apoptosis-inducing drugs are a mainstay of anticancer treatment regimens. The fields of pharmacology and toxicology consequently have a long history of investigating apoptotic cell death in the context of drug safety and efficacy studies. Canonical apoptotic cell death is crucially dependent on type II cysteinyl aspartate-specific proteases (caspases), and their activation is therefore widely used as a marker for this cell death modality. Here we describe a flow cytometric method for noninvasive, highly sensitive and reproducible FRET-based ...
Source: Springer protocols feed by Pharmacology/Toxicology - December 31, 2015 Category: Drugs & Pharmacology Source Type: news

A Low-Cost Method for Tracking the Induction of Apoptosis Using FRET-Based Activity Sensors in Suspension Cells
Apoptosis, or programmed cell death, is a tightly regulated cellular event that plays an important role in both normal developmental processes and many pathological states. The induction of apoptosis is tightly regulated through the coordinated action of members of the caspase family of proteases. Here we discuss a relatively inexpensive protocol for monitoring the induction and progression of apoptosis using a genetically encoded fluorescence resonance energy transfer (FRET)-based biosensor of the executioner caspase, caspase-3, in living suspension cells. (Source: Springer protocols feed by Pharmacology/Toxicology)
Source: Springer protocols feed by Pharmacology/Toxicology - December 31, 2015 Category: Drugs & Pharmacology Source Type: news

Detecting Apoptosis, Autophagy, and Necrosis
There are many commercially available kits to identify specific types of cell death, but at the present time, there is no simple assay that can distinguish apoptosis, necrosis, and autophagy. Autophagy and apoptosis are highly conserved processes that maintain organism and cellular homeostasis. They are also prime targets for the design of tumor therapeutics. Apoptosis is a highly regulated process involved in removing unwanted or unhealthy cells. Autophagy is a metabolic process, in which proteins and organelles are targeted for degradation in the lysosome. Necrosis is initiated by external factors, such as toxins, infect...
Source: Springer protocols feed by Pharmacology/Toxicology - December 31, 2015 Category: Drugs & Pharmacology Source Type: news

Measurement of Apoptosis by Multiparametric Flow Cytometry
Apoptosis remains a critical phenomenon in cell biology, playing a regulatory role in virtually every tissue system. It is particular crucial in the immune system, ranging from immature immune cell development and selection to downregulation of the mature immune response. Apoptosis is a primary mechanism in the action of antitumor drugs, and is thus an important phenomenon in pharmacology, drug discovery, and toxicology. Flow cytometry is the primary technique for measuring apoptosis in suspension cells; many flow cytometry assays have been developed to measure the entire apoptotic process, from the earliest signal transdu...
Source: Springer protocols feed by Pharmacology/Toxicology - December 31, 2015 Category: Drugs & Pharmacology Source Type: news

Determining the Extent of Toxicant-Induced Apoptosis Using Concurrent Phased Apoptosis Assays
Apoptosis is a stage-dependent process exhibiting characteristic biochemical, molecular and morphological features that vary progressively through the apoptosis process. Apoptosis induced by toxicants may activate varied features of apoptosis to different extents and kinetics. Some of the features activated may occur transiently, while others may not occur in a cell system undergoing toxicant-induced apoptosis. Thus, the best approach for quantitating the extent of toxicant-induced apoptosis involves the utilization of a combination of assays focusing on different morphological, biochemical and molecular features of apopto...
Source: Springer protocols feed by Pharmacology/Toxicology - December 31, 2015 Category: Drugs & Pharmacology Source Type: news

Detection of Apoptosis: From Bench Side to Clinical Practice
Apoptosis or programmed cell death is implicated in several pathological conditions, such as cancer and neurodegenerative diseases. An increasing number of therapies are developed by targeting apoptosis signaling components to either induce or inhibit apoptosis in target cells. For these reasons, it is critical to develop appropriate analytical methods for the detection of apoptotic cell death in the context of monitoring relevant disease progression and therapeutic effects of clinical treatments (e.g., chemotherapy in cancer patients). This review provides an overview of the currently used methods for detection of apoptos...
Source: Springer protocols feed by Pharmacology/Toxicology - December 31, 2015 Category: Drugs & Pharmacology Source Type: news

An Overview of Apoptosis Methods in Toxicological Research: Recent Updates
Apoptosis is the most common form of programmed cell death. Apoptosis plays a critical role in many physiological functions, and its dysregulation is an underlying defect in various diseases, including cancer. In fact, many toxicants and chemotherapeutic drugs exert their mechanisms of action through modulation of the apoptosis process. Thus, interest in the apoptosis process, as well as the methods used to assess and quantify its various aspects has continued to spike. This chapter provides a brief overview of the apoptosis process, the most common apoptosis methods, and the principles upon which these methods function. F...
Source: Springer protocols feed by Pharmacology/Toxicology - December 31, 2015 Category: Drugs & Pharmacology Source Type: news

Liposomes in Apoptosis Induction and Cancer Therapy
Cancer is the leading cause of death with multiple obstacles in therapeutic arsenals employed to date. Apoptosis induction in cancer cells has hitherto been a prominent unresolved obstacle for a few decades. Liposomes with multiple merits were extensively employed to entrap several types of anticancer agents, biomolecules and imaging agents to achieve substantial therapeutic effect for various types of cancers. Multifunctional liposomes with enhanced biocompatible properties were designed to enhance the therapeutic effect. Despite the promising drug delivery strategies and significantly reduced toxicity of the liposomal fo...
Source: Springer protocols feed by Pharmacology/Toxicology - December 31, 2015 Category: Drugs & Pharmacology Source Type: news

Targeting Cancer Cell Death with Small Molecule Agents for Potential Therapeutics
Time has come to switch from morphological to molecular definitions of cell death subroutines, due to substantial progress in biochemical and genetic exploration. Currently, cell death subroutines are defined by a series of precise, measurable biochemical features; these include apoptosis, autophagic cell death and necroptosis. Accumulating evidence has gradually revealed the core molecular machinery of cell death in carcinogenesis; the intricate relationships between cell death subroutines and cancer, however, still need to be clarified. Cancer drug discovery, in particular, has benefitted significantly from a rapid progr...
Source: Springer protocols feed by Pharmacology/Toxicology - December 31, 2015 Category: Drugs & Pharmacology Source Type: news

Microplate-Based Whole Zebrafish Caspase 3/7 Assay for Screening Small Molecule Compounds
In this research, using a commercially available human specific caspase 3/7 chemiluminescent test kit (Caspase 3/7 Glo, Promega, Madison, WI), developed for cell based assays, we describe a microplate-based whole zebrafish assay format to identify potential small molecule caspase inhibitors and activators. Based on the high degree of evolutionary conservation among species, we show that human specific 3/7 substrate cross reacts with zebrafish. Using untreated zebrafish, optimum assay conditions (including substrate concentration, number of zebrafish per microwell, and incubation time to generate a linear reaction) are dete...
Source: Springer protocols feed by Pharmacology/Toxicology - December 31, 2015 Category: Drugs & Pharmacology Source Type: news

Novel Electrochemical Biosensor for Apoptosis Evaluation
Apoptosis evaluation is one of the most important tasks of toxicology. By using a peptide as the recognition element, and assembling apoptotic cells on a solid surface, we have established a novel electrochemical method for the detection of apoptosis levels. Such a peptide-based electrochemical biosensor is simple, cost-effective, convenient, and sensitive. Since the results obtained are well in line with other standard methods, this method holds a great potential towards the analysis of apoptosis and its applications. In this chapter, we introduce a general overview of this technical approach for detecting apoptotic cells...
Source: Springer protocols feed by Pharmacology/Toxicology - December 31, 2015 Category: Drugs & Pharmacology Source Type: news

Experiment-Guided Molecular Modeling of Protein & ndash;Protein Complexes Involving GPCRs
Experimental structure determination for G protein-coupled receptors (GPCRs) and especially their complexes with protein and peptide ligands is at its infancy. In the absence of complex structures, molecular modeling and docking play a large role not only by providing a proper 3D context for interpretation of biochemical and biophysical data, but also by prospectively guiding experiments. Experimentally confirmed restraints may help improve the accuracy and information content of the computational models. Here we present a hybrid molecular modeling protocol that integrates heterogeneous experimental data with force field-b...
Source: Springer protocols feed by Pharmacology/Toxicology - August 13, 2015 Category: Drugs & Pharmacology Source Type: news

The Dynamic Process of Drug & ndash;GPCR Binding at Either Orthosteric or Allosteric Sites Evaluated by Metadynamics
Major advances in G Protein-Coupled Receptor (GPCR) structural biology over the past few years have yielded a significant number of high-resolution crystal structures for several different receptor subtypes. This dramatic increase in GPCR structural information has underscored the use of automated docking algorithms for the discovery of novel ligands that can eventually be developed into improved therapeutics. However, these algorithms are often unable to discriminate between different, yet energetically similar, poses because of their relatively simple scoring functions. Here, we describe a metadynamics-based approach to ...
Source: Springer protocols feed by Pharmacology/Toxicology - August 13, 2015 Category: Drugs & Pharmacology Source Type: news

Use of Fluorescence Indicators in Receptor Ligands
Fluorescence techniques can provide insights into the environment of fluorescence indicators incorporated within a ligand as it is bound to its receptor. Fluorescence indicators of different sizes and chemical characteristics can provide insights into the nature of the binding environment, the surrounding structures, and even into conformational changes associated with receptor activation. Methods for determining fluorescence spectral analysis, fluorescence quenching, fluorescence anisotropy, fluorescence lifetimes, and red edge excitation shifts of the ligand probes are described. The applications of these techniques to t...
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news

Monitoring G Protein Activation in Cells with BRET
Live-cell assays based on fluorescence and luminescence are now indispensable tools for the study of G protein signaling. Assays based on fluorescence and bioluminescence resonance energy transfer (FRET and BRET) have been particularly valuable for monitoring changes in second messengers, protein–protein interactions, and protein conformation. Here, we describe a BRET assay that monitors the release of free Gβγ dimers after activation of heterotrimers containing Gα subunits from all four G protein subfamilies. This assay provides useful kinetic and pharmacological information with reasonably high thr...
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news

Approaches to Characterize and Quantify Oligomerization of GPCRs
Fluorescence resonance energy transfer (FRET) is an approach widely used to detect protein–protein interactions in live cells. This approach is based on the sensitization of an “acceptor” molecule by the energy transfer from a “donor” when there is an overlap between the emission spectrum of the “donor” and the excitation spectrum of the “acceptor” and close proximity between the two fluorophore species (in the region of 8 nm). Various methods exist to quantify FRET signals: here, we describe the application of homogeneous time-resolved FRET (htrFRET) combined with Tag-...
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news

Quantitative Multi-color Detection Strategies for Bioorthogonally Labeled GPCRs
We describe multiple bioorthogonal approaches to label G protein-coupled receptors (GPCRs) heterologously expressed in mammalian cells. The use of genetically encoded unnatural amino acids as bioorthogonal tags results in receptors that are expressed at lower levels than even their low abundance wild-type counterparts. Therefore, reproducible and sensitive quantification of the labeled GPCRs is extremely important and conventional methods are simply not sufficiently accurate and precise. Silver stains lack reproducibility, spectroscopic methods using fluorescent ligands are limited to quantifying only functional receptor m...
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news

Single-Molecule Fluorescence Microscopy for the Analysis of Fast Receptor Dynamics
Assessing the dynamics of individual membrane proteins in living cells is a powerful approach to investigate their assembly, mobility, and function. Here, we describe how to image single G protein-coupled receptors (GPCRs), both in the active and inactive state. This is achieved by combining labeling of GPCRs with bright organic fluorophores and fluorescent imaging by total internal reflection fluorescence microscopy. Using this method, individual tracks of single molecules can be analyzed in parallel with high spatial precision and with frame rates up to 50/s. (Source: Springer protocols feed by Pharmacology/Toxicology)
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news

Nuts and Bolts of CF3 and CH3 NMR Toward the Understanding of Conformational Exchange of GPCRs
With the advent of efficient protein expression and functional purification protocols, it is now possible to reconstitute many G protein-coupled receptors (GPCRs) in detergent micelles at concentrations of 25 μM or more. Such concentrations are sufficient for studies of conformational states and dynamics relating to function and the mechanism of activation of GPCRs, using solution state NMR. In particular, methyl spectroscopy, in the form of one-dimensional 19F NMR or two-dimensional (1H,13C) NMR, provides high fidelity spectra which reveal detailed features associated with conformational states and their lifetimes, as ...
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news

2D Projection Analysis of GPCR Complexes by Negative Stain Electron Microscopy
While electron cryo-microscopy (cryo-EM) of biological specimens is the preferred single particle EM method for structure determination, its application is very challenging for the typically small (
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news

Purification and Crystallization of a Thermostabilized Agonist-Bound Conformation of the Human Adenosine A2A Receptor
Crystallization of G protein-coupled receptors (GPCRs) is successful due to the development of generic protein engineering strategies, which has resulted in the structure determination of more than 25 GPCRs, including representatives from class A, B, C, and F. Most of the X-ray structures available correspond to an inactive conformation of the receptor bound to an antagonist. Only a few high-resolution structures of agonist-bound conformations of GPCRs have been determined over the last 6 years. Here, we describe the purification and crystallization protocols of a thermostabilized agonist-bound conformation of the human ad...
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news

Interaction Fingerprints and Their Applications to Identify Hot Spots
Binding recognition is in the core of how nature controls processes in living cells, how enzyme–substrate binding leads to catalysis and how drugs modulate enzymes and receptors to convey a desirable physiological response. Thus, understanding binding recognition in a systematic manner is paramount, not only to understand biological processes but also to be able to design and discover new bioactive compounds. One such way to analyze binding interactions is through the development of binding interaction fingerprints. Here, we present the methodology to develop interaction fingerprints with three different software pla...
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news

Purification of Stabilized GPCRs for Structural and Biophysical Analyses
G protein-coupled receptors (GPCRs) are of particular importance for drug discovery, being the targets of many existing drugs, and being linked to many diseases where new therapies are required. However, as integral membrane proteins, they are generally unstable when removed from their membrane environment, precluding them from the wide range of structural and biophysical techniques which can be applied to soluble proteins such as kinases. Through the use of protein engineering methods, mutations can be identified which both increase the thermostability of GPCRs when purified in detergent, as well as biasing the receptor t...
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news

Experiment-Guided Molecular Modeling of Protein–Protein Complexes Involving GPCRs
Experimental structure determination for G protein-coupled receptors (GPCRs) and especially their complexes with protein and peptide ligands is at its infancy. In the absence of complex structures, molecular modeling and docking play a large role not only by providing a proper 3D context for interpretation of biochemical and biophysical data, but also by prospectively guiding experiments. Experimentally confirmed restraints may help improve the accuracy and information content of the computational models. Here we present a hybrid molecular modeling protocol that integrates heterogeneous experimental data with force field-b...
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news

The Dynamic Process of Drug–GPCR Binding at Either Orthosteric or Allosteric Sites Evaluated by Metadynamics
Major advances in G Protein-Coupled Receptor (GPCR) structural biology over the past few years have yielded a significant number of high-resolution crystal structures for several different receptor subtypes. This dramatic increase in GPCR structural information has underscored the use of automated docking algorithms for the discovery of novel ligands that can eventually be developed into improved therapeutics. However, these algorithms are often unable to discriminate between different, yet energetically similar, poses because of their relatively simple scoring functions. Here, we describe a metadynamics-based approach to ...
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news

Docking and Virtual Screening Strategies for GPCR Drug Discovery
Progress in structure determination of G protein-coupled receptors (GPCRs) has made it possible to apply structure-based drug design (SBDD) methods to this pharmaceutically important target class. The quality of GPCR structures available for SBDD projects fall on a spectrum ranging from high resolution crystal structures (3 Å) where some protein residues are not resolved, and finally to homology models that are built using distantly related templates. Each GPCR project involves a distinct set of opportunities and challenges, and requires different approaches to model the interaction between the receptor and the ligan...
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news

Radioligand Binding Assay for an Exon 11-Associated Mu Opioid Receptor Target
Receptor binding provides a valuable approach for characterization of drugs and their receptors. There are three major families of opioid receptors: mu, delta, and kappa. Highly selective radioligands are available for all three classes of traditional receptors. Of the three, the mu receptor undergoes extensive alternative splicing, generating a number of traditional mu receptor subtypes as well as a nontraditional, truncated set of variants associated with exon 11. These exon 11-associated truncated variants are not readily labeled with current radioligands. Here we describe the synthesis of a radioiodinated ligand suitab...
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news

High-Throughput Screening for Allosteric Modulators of GPCRs
We describe several HTS protocols designed for the identification of GPCR ligands, with a particular focus on the identification of allosteric modulators. (Source: Springer protocols feed by Pharmacology/Toxicology)
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news

Virus-Mediated Expression of DREADDs for In Vivo Metabolic Studies
During the past few years, CNO-sensitive designer G protein-coupled receptors (GPCRs) known as DREADDs (designer receptors exclusively activated by designer drugs) have emerged as powerful new tools for the study of GPCR physiology. In this chapter, we present protocols employing adeno-associated viruses (AAVs) to express a Gq-coupled DREADD (Dq) in two metabolically important cell types, AgRP neurons of the hypothalamus and hepatocytes of the liver. We also provide examples dealing with the metabolic analysis of the Dq mutant mice after administration of CNO in vivo. The approaches described in this chapter can be applied...
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news

Bioluminescence Resonance Energy Transfer Approaches to Discover Bias in GPCR Signaling
Bioluminescence resonance energy transfer (BRET) is a well-established technique for investigating G protein-coupled receptor (GPCR) pharmacology. BRET enables the monitoring of molecular proximity through the use of heterologously expressed proteins of interest and/or fluorophore-labeled ligands. Fusion to a donor luciferase enzyme or an acceptor fluorophore and subsequent detection of resonance energy transfer indicate the close proximity of the molecules of interest. As BRET is readily applied to the study of numerous GPCR signaling and regulatory paths, it is an ideal technique for investigating the pharmacology of bia...
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news

Approaches to Assess Functional Selectivity in GPCRs: Evaluating G Protein Signaling in an Endogenous Environment
This report presents our method and offers tips for evaluating G protein signaling in endogenous tissues. Predominately, brain tissues are discussed here; optimization points that can be applied to any tissues are highlighted. (Source: Springer protocols feed by Pharmacology/Toxicology)
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news

The Measurement of Receptor Signaling Bias
This chapter describes a method to quantify biased signaling effects of agonists; this approach can furnish a scale for medicinal chemists to optimize biased profiles. Biased ligands have different pharmacological properties on a molecular level (stabilization of different receptor active states) and thus can have different pharmacological profiles therapeutically. The calculation of transduction ratios (ΔΔlog(τ/K A)) values (where τ is efficacy and K A a measure of affinity) allows the identification of agonists that demonstrate unique signaling either for different signaling pathways linked to the rec...
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news

Detection and Quantification of Intracellular Signaling Using FRET-Based Biosensors and High Content Imaging
Förster resonance energy transfer (FRET) biosensors represent invaluable tools to detect the spatiotemporal context of second messenger production and intracellular signaling that cannot be attained using traditional methods. Here, we describe a detailed protocol for the use of high content imaging in combination with FRET biosensors to assess second messenger production and intracellular signaling in a time-effective manner. We use four different FRET biosensors to measure cAMP levels, kinase (ERK and PKC), and GTPase activity. Importantly, we provide the protocols to express and measure these sensors in a variety of...
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news

A Systems Toxicology Approach to Investigating the Cardiovascular Effects of Cigarette Smoke and Environmental Pollutants in ApoE-Deficient Mice
Epidemiological evidence indicates that exposure to combustion-derived particles is linked to an increased risk of cardiovascular disease. Despite this strong association, there remains a lack of data that can be used to identify the molecular mechanisms through which exposure to particulate matter (PM) leads to adverse cardiovascular events. The biological complexity of the responses generated by exposure to PM is compounded by the inherent multidimensional nature of the chemical mixtures associated with PM emitted from a range of sources, including diesel and gasoline exhausts, cigarette smoke, and ambient particles. The...
Source: Springer protocols feed by Pharmacology/Toxicology - July 23, 2015 Category: Drugs & Pharmacology Source Type: news

High-Content Screening: Understanding and Managing Mechanistic Data to Better Predict Toxicity
An increased understanding of the cellular pathways involved in toxicity responses, coupled with a simultaneous advance in technology, has allowed for a shift in the way that the safety assessment of novel chemicals is performed. The development of assays that offer a high-throughput and low-cost option in comparison to more traditional approaches has been a focus of recent years. (Source: Springer protocols feed by Pharmacology/Toxicology)
Source: Springer protocols feed by Pharmacology/Toxicology - July 23, 2015 Category: Drugs & Pharmacology Source Type: news

Xenobiotic Metabolism Activation as a Biomarker of Cigarette Smoke Exposure Response
The recent advances in “omics” technologies have generated various in silico approaches for toxicity assessment. In silico-based toxicity predictions can overcome certain major drawbacks of laboratory experiments, including the limitation of conducting experiments in a chemical-by-chemical basis that can be expensive. This chapter discusses some recent applications of in silico approaches utilizing xenobiotic metabolism that can be used to assess the impact of cigarette smoke (CS). We first outline recent studies using quantum mechanics/molecular modeling and quantitative structure–activity relationships ...
Source: Springer protocols feed by Pharmacology/Toxicology - July 23, 2015 Category: Drugs & Pharmacology Source Type: news

Analysis of Proteomic Data for Toxicological Applications
Toward a comprehensive characterization of toxicant responses, systems toxicology requires the integration of different data modalities. Proteomics approaches measure changes in the levels of proteins and their posttranslational modifications, which can closely reflect the biological effects of a toxicant. With a focus on mass spectrometry-based proteomics, we describe the isobaric tag-based approach for quantitative proteomics (iTRAQ® and TMT™) and describe computational approaches to derive biological/mechanistic insights. Specifically, we describe the generation and quantification of mass-spectrometry data and...
Source: Springer protocols feed by Pharmacology/Toxicology - July 23, 2015 Category: Drugs & Pharmacology Source Type: news

Quantifying the Biological Impact of Active Substances Using Causal Network Models
In this chapter a five-step strategy is described that provides comparative evaluations of the effects of biologically active substances. These evaluations constitute an integral part of the determination of the risks for the human population to exposure to these substances. The strategy is based on the concept of biological impact quantification for which novel computational methodologies have been developed in the past few years; these methodologies are reviewed in this chapter. The effects of the active substances are then described in terms of networks containing the biological mechanisms involved in the response to th...
Source: Springer protocols feed by Pharmacology/Toxicology - July 23, 2015 Category: Drugs & Pharmacology Source Type: news

Mitochondrial Targeting of Catalytic RNAs
We describe here mitochondrial targeting of trans-cleaving ribozymes destined to knockdown organelle RNAs for regulation studies and inverse genetics and biotechnological purposes. The design and functional assessment of chimeric RNAs combining the ribozyme and the mitochondrial shuttle are detailed, followed by all procedures to prepare constructs for in vivo expression, generate stable plant transformants, and establish target RNA knockdown in mitochondria. (Source: Springer protocols feed by Pharmacology/Toxicology)
Source: Springer protocols feed by Pharmacology/Toxicology - February 5, 2015 Category: Drugs & Pharmacology Source Type: news

Mitochondrial Targeting of Recombinant RNA
Mitochondrial import of small noncoding RNA is found in a large variety of species. In mammalian cells, this pathway can be used for therapeutic purpose, to restore the mitochondrial functions affected by pathogenic mutations. Recently, we developed mitochondrial RNA vectors able to address therapeutic oligoribonucleotides into human mitochondria. Here we provide the protocol for transfection of cultured human cells with small recombinant RNA molecules and describe two approaches useful to demonstrate their import into mitochondria: (1) isolation of RNA from purified mitochondria and quantitative hybridization analysis and...
Source: Springer protocols feed by Pharmacology/Toxicology - February 5, 2015 Category: Drugs & Pharmacology Source Type: news

Allotopic Expression of ATP6 in the Mouse as a Transgenic Model of Mitochondrial Disease
Progress in animal modeling of polymorphisms and mutations in mitochondrial DNA (mtDNA) is not as developed as nuclear transgenesis due to a host of cellular and physiological distinctions. mtDNA mutation modeling is of critical importance as mutations in the mitochondrial genome give rise to a variety of pathological conditions and play a contributing role in many others. Nuclear localization and transcription of mtDNA genes followed by cytoplasmic translation and transport into mitochondria (allotopic expression, AE) provide an opportunity to create in vivo modeling of a targeted mutation in mitochondrial genes and has b...
Source: Springer protocols feed by Pharmacology/Toxicology - February 5, 2015 Category: Drugs & Pharmacology Source Type: news

Analysis of Pollutant-Induced Changes in Mitochondrial DNA Methylation
There is increasing evidence that exposure to air pollutants is associated with human disease and may act through epigenetic modification of the nuclear genome, but there have been few publications describing their impact upon the mitochondrial genome. Mitochondrial DNA may be more susceptible to pollutant-induced changes via increased oxidative stress in the cell, and therefore this field of research is of growing interest. Many techniques employed to study DNA methylation of the nuclear genome are also applicable to mitochondrial epigenetic studies. In this chapter, we describe a protocol for the isolation of mitochondri...
Source: Springer protocols feed by Pharmacology/Toxicology - February 5, 2015 Category: Drugs & Pharmacology Source Type: news

ETS and DEAS Studies of the Reduction of Xenobiotics in Mitochondrial Intermembrane Space
This chapter describes the complementary experimental techniques electron transmission spectroscopy (ETS) and dissociative electron attachment spectroscopy (DEAS), two of the most suitable means for investigating interactions between electrons and gas-phase molecules, resonance formation of temporary molecular negative ions, and their possible decay through the dissociative electron attachment (DEA) mechanism. The latter can be seen as the gas-phase counterpart of the transfer of a solvated electron in solution, accompanied by dissociation of the molecular anion, referred to as dissociative electron transfer (DET). DET tak...
Source: Springer protocols feed by Pharmacology/Toxicology - February 5, 2015 Category: Drugs & Pharmacology Source Type: news

DQAsomes as the Prototype of Mitochondria-Targeted Pharmaceutical Nanocarriers: Preparation, Characterization, and Use
DQAsomes (dequalinium-based liposome-like vesicles) are the prototype for all mitochondria-targeted vesicular pharmaceutical nanocarrier systems. First described in 1998, they have been successfully explored for the delivery of DNA and low-molecular weight molecules to mitochondria within living mammalian cells. Potential areas of application involve mitochondrial gene therapy, antioxidant therapy as well as apoptosis-based anticancer chemotherapy. Here, detailed protocols for the preparation, characterization, and application of DQAsomes are given. (Source: Springer protocols feed by Pharmacology/Toxicology)
Source: Springer protocols feed by Pharmacology/Toxicology - February 5, 2015 Category: Drugs & Pharmacology Source Type: news

Analysis of Mitochondrial Morphology and Function Under Conditions of Mitofusin 2 Deficiency
Recent discoveries linking mitochondrial dynamics to various pathologies have generated interest in the design of robust methods to screen proteins involved in mitochondrial dynamics and small molecules that modulate mitochondrial dynamics. Here, we describe functional screening protocols to analyze mitochondrial parameters such as mitochondrial morphology, reactive oxygen species (ROS) levels, mitochondrial calcium, and oxygen consumption rate in cultured cells. Novel proteins that participate in the regulation of mitochondrial dynamics and function can be identified using such an approach. (Source: Springer protocols fee...
Source: Springer protocols feed by Pharmacology/Toxicology - February 5, 2015 Category: Drugs & Pharmacology Source Type: news

Yeast as a Tool to Study Mitochondrial Retrograde Pathway En Route to Cell Stress Response
Mitochondrial retrograde signaling is a mitochondria-to-nucleus communication pathway, conserved from yeast to humans, by which dysfunctional mitochondria relay signals that lead to cell stress adaptation in physiopathological conditions by changes in nuclear gene expression. The best comprehension of components and regulation of retrograde signaling have been obtained in Saccharomyces cerevisiae, where retrograde target gene expression is regulated by RTG genes. In this chapter, we describe the methods to measure mitochondrial retrograde pathway activation in yeast cells by monitoring the mRNA levels of RTG target genes, ...
Source: Springer protocols feed by Pharmacology/Toxicology - February 5, 2015 Category: Drugs & Pharmacology Source Type: news

Cell Energy Budget Platform for Assessment of Cell Metabolism
We describe a simple methodology for high-throughput multiparametric assessment of cell bioenergetics, called cell energy budget (CEB) platform, and demonstrate its practical use with cell models. The CEB relies on a standard multi-label reader with time-resolved fluorescence capabilities, the lanthanide probe pH-Xtra™ to measure extracellular acidification (ECA) associated with lactate (L-ECA) and combined lactate/CO2 (T-ECA) extrusion, the phosphorescent probe MitoXpress®-Xtra to measure oxygen consumption rate (OCR), the bioluminescent total ATP assay, and absorbance-based total protein assay. This approach ca...
Source: Springer protocols feed by Pharmacology/Toxicology - February 5, 2015 Category: Drugs & Pharmacology Source Type: news

In Vivo Visualization and Quantification of Mitochondrial Morphology in C. elegans
Caenorhabditis elegans is a highly malleable model system, intensively used for functional, genetic, cytometric, and integrative studies. Due to its simplicity and large muscle cell number, C. elegans has frequently been used to study mitochondrial deficiencies caused by disease or drug toxicity. Here, we describe a robust and efficient method to visualize and quantify mitochondrial morphology in vivo. This method has many practical and technical advantages above traditional (manual) methods and provides a comprehensive analysis of mitochondrial morphology. (Source: Springer protocols feed by Pharmacology/Toxicology)
Source: Springer protocols feed by Pharmacology/Toxicology - February 5, 2015 Category: Drugs & Pharmacology Source Type: news